Interpretive Summary: The goal of effective nematode control is to protect food animals from production losses by reducing parasite transmission and the rate of parasite establishment in the host. Accurate and rapid identification of species infecting the host is essential to achieving these goals. We previously developed a rapid and sensitive DNA test to diagnose gastrointestinal (GI) nematode eggs from ruminant feces as well as methods for increasing the yield, purity and speed of DNA isolation from eggs. However, this technology has yet to be adapted to real-time PCR, currently, one of the fastest growing technologies for gene identification and quantitation. Herein we evaluate a matrix of isolation and purification parameters for applicability to multiplex real-time PCR of fecal eggs to enhance the sensitivity, speed and objectivity of the identification test. Such a test will lend itself to automated analyses and permit more selective drug treatment of animals in order to reduce on-farm drug resistance developing in parasites, reduce the potential for harmful drug residues to collect in animals, and reduce costs to the producer groups by identifying and treating only those animals harboring pathogenic parasites. Application of these methods extends well beyond use with GI nematodes of cattle.

Technical Abstract:
The study was performed to determine the feasibility of using real-time PCR for quantifying feces-derived trichostrongyle eggs, a technology not yet reported for this stage. Haemonchus contortus eggs were used to evaluate fecal contaminants, time following egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from 5-75 eggs. However, threshold cycle (Ct) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in Ct values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 hrs. Non-competitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at 10-fold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs. It further outlines potential limitations supporting the need for multiplex assays over simplex assays.